Display technologies have been developed to combine information storage and amplification capabilities of nucleic acids with the functional activities of other compound. Display technologies rely on an association between a functional entity and a nucleic acid sequence informative about the structure of the functional entity. An advantage of such methods is that very large libraries can be constructed and probed for a desired activity of the functional entities. Library members having the desired activity can then be partitioned from library members not having the desired activity, thus creating an enriched library with a higher fraction of members having the desired activity. This process is called selection. The structures of the library members in the enriched library can then be identified by their cognate nucleic acid sequence, thus allowing identification even from minute amounts of material.
Some display technologies further allow the enriched library to be amplified without knowing the identity of its members; not merely the nucleic acid sequences but the functional entities too. Such display technologies are called “amplifiable display technologies”. These technologies are especially advantageous when dealing with large libraries, because iterative rounds of selection and amplification can be performed allowing increased enrichment of desired activities. Another advantage of amplifiable display technologies is that rounds of selection, amplification and diversification can be performed, thus using the same principle as in natural selection, to evolve molecules with desired function. This process is called molecular evolution.
Display technologies utilizing biological systems have been developed, the most notable of which is phage display (Smith, Science, 228, 1315-7, 1985). However, such systems are limited to the display of natural occurring products such as proteins and peptides.
In vitro display technologies exploiting the flexibility of organic chemistry has been described. One example is described in U.S. Pat. No. 5,723,598. The method uses a bifunctional molecule; one functionality capable of accepting a chemical group and one functionality capable of accepting a nucleic acid sequence. The method for synthesizing such a library is commonly known as “split and mix”, consisting of rounds of mixing and splitting the bifunctional molecules into compartments. A compartment specific pair of chemical group and nucleic acid sequence is added. The nucleic acid sequences are thus encoding the chemical groups. All bifunctional molecules are then mixed and the process iterated to create a large combinatorial library. The library is then subjected to selection and the selected nucleic acid sequences amplified by PCR, which may be used for identification by conventional molecular biology; cloning and DNA sequencing.
Another example is described in WO04/039825A2, where a combinatorial library is created, by rounds of proximity-guided addition of cognate pairs of chemical group and nucleic acid code to a bifunctional molecule; one functionality capable of accepting a chemical group and one functionality capable of accepting a nucleic acid sequence. Furthermore repertoires of so-called transfer units are used, where a chemical group is attached to an oligonucleotide, containing a coding segment and a segment capable of annealing to the bifunctional molecule. A repertoire of transfer units is annealed to the bifunctional molecules, which allows the code to be transferred enzymatically to the bifunctional molecule, as well as guiding the chemical group to react with the very same bifunctional molecule. This process can be reiterated to create a large combinatorial library.
The libraries described above can be subjected to selections to form an enriched library. The enriched libraries members' synthetic history can subsequently be deduced through the encoding nucleic acid. A limitation of these approaches is however that the enriched library cannot be amplified.
In vitro display technologies taking advantage of the flexibility of organic chemistry and rounds of selections, amplification and diversification has been described. These methods rely on the use of templates.
One example is described in WO00/23458, using a “split and mix’ principle. A library of ssDNA templates is used, each containing a chemical reaction site and several positions of codon segments. The templates are compartmentalized by virtue of hybridizing to a repertoire of anti-codon sequences for a given codon position. Then, a compartment specific chemical reaction is performed modifying the reaction site on the templates. The templates are then mixed and the process reiterated by using other codon positions to form a combinatorial library.
Another example is described in WO02/074929A2, using a “single-pot” principle. A library of oligonucleotide templates is used, each containing a chemical reaction site and several positions of codon segments. Furthermore, using a repertoire of transfer units, the method consists of an oligonucleotide anti-codon sequence and a chemical reactive group. The library of templates are hybridized with a repertoire of transfer units for a given codon position. This brings the chemical group on a hybridized transfer unit in proximity to the reaction site on the hybridized template, which consequently guides the chemical reaction of cognate pairs. This process is then reiterated using other codon positions to form a combinatorial library.
A limitation of the above proximity guiding of cognate pairs of code and chemical group is given by the linear structure of the template oligonucleotide. As a consequence of the linearity the distance between codon and the chemical reaction site will differ from codon position to codon position. For codon positions longer away from the reaction site the proximity guiding becomes compromised, as the local concentration drops to the power of three as a function of the distance. This disadvantage becomes more pronounced for complex libraries, with more codon positions and more complex codons.
This problem is sought solved in WO04/016767A2, where the transfer units besides from an anti-codon segment also contain a constant segment, which is complementary to a constant sequence on the template close to the reactive site. Thus, by hybridizing a transfer unit to a template results in that the template sequence between the codon position in question and the constant segment is bulging out, to form a so-called omega structure. The concept is that the codon segment is responsible for the specificity and the constant segment responsible for the proximity. Also suggested in WO04/016767A2, is a so-called T-architecture of the templates, where the reactive site on the template is situated in the middle of the template, with the codon positions spread out on each side. Consequently, the distance problem is so called “cut in half”.
WO 2004/056994A2 discloses a method similar to WO02/074929A2 or WO04/016767A2 with the difference that the template is cut into minor sequences, termed connecting polynucleotides in the application. The connecting polynucleotides connect transfer units to bring these into reaction proximity. In certain embodiments the connecting polynucleotides may comprise a reactive chemical group. To obtain an encoded molecule the method is dependent upon codon/anti-codon recognition prior to reaction.
The template directed libraries described above are subsequently subjected to selections to form enriched libraries. The enriched libraries members' synthetic history can then be deduced through the encoding nucleic acid. The enriched libraries can also be amplified and diversified by for example error prone PCR, thus allowing for molecular evolution.
A limitation in these approaches is that a large number of templates have to be created, which is cumbersome, as the templates have to be of considerable length to ensure proper codon/anti-codon hybridization. In methods using a plurality of minor sequences to make up the final directed synthesis the number of sequences to be synthesized is even higher than the actual library size.
The prior art methods using templates suffer from the disadvantage that the encoding is dependent upon hybridization of codon and anti-codon sequences. Sometimes hybridization between single stranded oligonucleotides will happen without perfect complementarities. In the case of library construction the result is loss of the association between the encoding and the synthetic history. Consequently, upon selection positive codes may be de-selected and negative codes may be selected. For more complex libraries this disadvantage becomes more significant as the complexity of the single stranded oligonucleotides also increases, both with respect to numbers, length and sequences. This makes the processes more difficult to control.
As described above in vitro display technologies allowing display of a variety of compound classes, selections, amplifications and diversifications have been developed. However, there is still an ongoing need for improvement, especially with respect to the quality in library construction and of diversification. The present invention offers a method for producing an encoded molecule in which the method does not require the pre-synthesis of a large number of templates. Furthermore, the present method is not dependent upon codon/anti-codon recognition for an encoded molecule to be formed.